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1.
Mortimer M. Civan Kim Peterson-Yantorno Thomas G. O'Brien 《The Journal of membrane biology》1987,97(3):193-204
Summary The phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate) stimulates baseline Na+ transport across frog skin epithelium and partially inhibits the natriferic response to vasopressin. The effects are produced largely or solely when TPA is added to the mucosal surface of the tissue. Although TPA activates protein kinase C, it has other effects, as well. Thus, the biochemical basis for the effects and the ionic events involved have been unclear. Furthermore, the physiologic implications have been obscure because of the sidedness of TPA's actions.We now report that two synthetic diacylglycerols (DAG) replicate the stimulatory and inhibitory effects of TPA on frog skin. DAG is the physiologic activator of PKC. In this tissue, it produces half-maximal stimulation at a concentration of 19 M. In contrast to TPA, DAG is about equally effective from either tissue surface.In a series of eight experiments, DAG was found to depolarize the apical membrane. Diacylglycerol also increases the paracellular conductance of frog skins bathed with mucosal Cl– Ringer's solution. The latter effect can be minimized by replacing NO
3
–
for Cl– in the mucosal solution. Under these conditions, combined intracellular and transepithelial measurements indicated that DAG increased both the apical Na+ permeability and intracellular Na+ concentration. These results are qualitatively similar to the effects of cyclic 3, 5-AMP on this tissue, suggesting that activation of PKC by DAG causes phosphorylation of the same or nearby gating sites phosphorylated by cAMP.We propose that apical Na+ entry is regulated in part by activation of PKC, and that insulin may be a physiologic trigger of this activation. 相似文献
2.
Summary The effect of papaverine, an inhibitor of the phosphodiesterase responsible for breakdown of cAMP, on the transepithelial sodium transport across the isolated frog skin was investigated.Serosal addition of papaverine caused initially an increase in the short-circuit current (SCC), a doubling of the cellular cAMP content and a depolarization of the intracellular potential under SCC conditions (V
scc).The initial increase in the SCC was followed by a pronounced decrease both in the SCC and in the natriferic action of antidiuretic hormone (ADH), but papaverine had no inhibitory effect on the ability of ADH to increase the cellular cAMP content. As SCC declines, no hyperpolarization was observed.The I/V relationship across the apical membrane during the inhibitory phase, revealed that papaverine reduces the sodium permeability of the apical membrane (P
Na
a
)as well as intracellular sodium concentration. These observations and the previously noted effect of papaverine on V
scc indicates that papaverine must have an effect on the cellular Cl or K permeability.The basolateral Na,K,2Cl cotransporter was blocked with bumetanide, which should bring the cellular chloride in equilibrium. Bumetanide had no effect on basal SCC and V
scc. When papaverine was added to skins preincubated with bumetanide, the effect of papaverine on SCC and V
scc was unchanged. Therefore, the depolarization of V
scc, observed during the papaverine induced inhibition of the SCC, must be due to a reduction in the cellular K permeability.In conclusion, it is suggested that papaverine reduces the sodium permeability of the apical membrane and the potassium permeability of the basolateral membrane of the frog skin epithelium. 相似文献
3.
4.
Summary In studies of apical membrane current-voltage relationships, in order to avoid laborious intracellular microelectrode techniques, tight epithelia are commonly exposed to high serosal K concentrations. This approach depends on the assumptions that high serosal K reduces the basolateral membrane resistance and potential to insignificantly low levels, so that transepithelial values can be attributed to the apical membrane. We have here examined the validity of these assumptions in frog skins (Rana pipiens pipiens). The skins were equilibrated in NaCl Ringer's solutions, with transepithelial voltageV
t clamped (except for brief perturbations V
t) at zero. The skins were impaled from the outer surface with 1.5m KCl-filled microelectrodes (R
el>30 M). The transepithelial (short-circuit) currentl
i and conductanceg
t=–I
t/V
t, the outer membrane voltageV
o (apical reference) and voltage-divider ratio (F
o=V
o/V
t), and the microelectrode resistanceR
el were recorded continuously. Intermittent brief apical exposure to 20 m amiloride permitted estimation of cellular (c) and paracellular (p) currents and conductances. The basolateral (inner) membrane conductance was estimated by two independent means: either from values ofg
i andF
o before and after amiloride or as the ratio of changes (–I
c/V
i) induced by amiloride. On serosal substitution of Na by K, within about 10 min,I
c declined andg
t increased markedly, mainly as a consequence of increase ing
p. The basolateral membrane voltage (V
i(=–V
o) was depolarized from 75±4 to 2±1 mV [mean±sem (n=6)], and was partially repolarized following amiloride to 5±2 mV. The basolateral conductance increased in high serosal K, as estimated by both methods. Essentially complete depolarization of the basolateral membrane and increase in its conductance in response to high [K] were obtained also when the main serosal anion was SO4 or NO3 instead of Cl. On clampingV
t over the range 0 to +125 mV in K2SO4-depolarized skins, the quasi-steady-stateV
o
V
t relationship was linear, with a mean slope of 0.88±0.03. The above results demonstrate that, in a variety of conditions, exposure to high serosal K results in essentially complete depolarization of the basolateral membrane and a large increase in its conductance. 相似文献
5.
Bent Vestergaard-Bogind Per Stampe Palle Christophersen 《The Journal of membrane biology》1985,87(1):67-75
Summary We have examined the effect of internal and external pH on Na+ transport across toad bladder membrane vesicles. Vesicles prepared and assayed with a recently modified procedure (Garty & Asher, 1985) exhibit large, rheogenic, amiloridesensitive fluxes. Of the total22Na uptake measured 0.5–2.0 min after introducing tracer, 80±4% (mean±se,n=9) is blocked by the diuretic with aK
1
of 2×10–8
m. Thus, this amiloridesensitive flux is mediated by the apical sodium-selective channels. Varying the internal (cytosolic) pH over the physiologic range 7.0–8.0 had no effect on sodium transport; this result suggests that variation of intracellular pHin vivo has no direct apical effect on modulating sodium uptake. On the other hand,22Na was directly and monotonically dependent on external pH. External acidification also reduced the amiloride-sensitive efflux across the walls of the vesicles. This inhibition of22Na efflux was noted at external Na+ concentrations of both 0.2 m and 53mm.These results are different from those reported with whole toad bladder. A number of possible bases for these differences are considered and discussed. We suggest that the natriferic response induced by mucosal acidification of whole toad urinary bladder appears to operate indirectly through one or more factors, presumably cytosolic, present in whole cells and absent from the vesicles. 相似文献
6.
Katsumasa Kawahara 《The Journal of membrane biology》1985,88(3):283-292
Summary The apical membrane K+ permeability of the newt proximal tubular cells was examined in the doubly perfused isolated kidney by measuring the apical membrane potential change (V
a
change) during alteration of luminal K+ concentration and resultant voltage deflections caused by current pulse injection into the lumen.V
a
change/decade for K+ was 50 mV at K+ concentration higher than 25mm, and the resistance of the apical membrane decreased bt 58% of control when luminal K+ concentration was increased from 2.5 to 25mm. Ba2+ (1mm in the lumen) reducedV
a
change/decade to 24 mV and increased the apical membrane resistance by 70%. These data support the view that Ba2+-sensitive K+ conductance exists in the apical membrane of the newt proximal tubule. Furthermore, intracellular K+ activity measured by K+-selective electrode was 82.4 ± 3.6 meq/liter, which was higher than that predicted from the Nernst equation for K+ across both cell membranes. Thus, it is concluded that cell K+ passively diffuses, at least in part, through the K+ conductive pathway of the apical membrane. 相似文献
7.
Intracellular calcium content of human erythrocytes: Relation to sodium transport systems 总被引:2,自引:0,他引:2
Summary To study the possible role of intracellular Ca (Ca
i
) in controlling the activities of the Na+–K+ pump, the Na+–K+ cotransport and the Na+/Li+ exchange system of human erythrocytes, a method was developed to measure the amount of Ca embodied within the red cell. For complete removal of Ca associated with the outer aspect of the membrane, it proved to be essential to wash the cells in buffers containing less than 20nm Ca. Ca was extracted by HClO4 in Teflon® vessels boiled in acid to avoid Ca contaminations and quantitated by flameless atomic absorption. Ca
i
of fresh human erythrocytes of apparently healthy donors ranged between 0.9 and 2.8 mol/liter cells. The mean value found in females was significantly higher than in males. The interindividual different Ca contents remained constant over periods of more than one year. Sixty to 90% of Ca
i
could be removed by incubation of the cells with A23187 and EGTA. The activities of the Na+–K+ pump, of Na+–K+ cotransport and Na+/Li+ exchange and the mean cellular hemoglobin content fell with rising Ca
i
; the red cell Na+ and K+ contents rose with Ca
i
. Ca depletion by A23187 plus EGTA as well as chelation of intracellular Ca2+ by quin-2 did not significantly enhance the transport rates. It is concluded that the large scatter of the values of Ca
i
of normal human erythrocytes reported in the literature mainly results from a widely differing removal of Ca associated with the outer aspect of the membrane. 相似文献
8.
Wenhui Wang Robert M. Henderson John Geibel Stanley White Gerhard Giebisch 《The Journal of membrane biology》1989,112(3):277-289
Summary Isolated early distal tubule cells (EDC) of frog kidney were incubated for 20–28 hr in the presence of aldosterone and then whole-cell K+ currents were measured at constant intracellular pH by the whole-cell voltage-clamp technique. Aldosterone increased barium-inhibitable whole-cell K+ conductance (gK+) threefold. This effect was reduced by amiloride and totally abolished by ouabain. However, aldosterone could still raisegK+ in ouabain-treated cells in the presence of furosemide.We tested whether changes in intracellular pH (pH
i
) could be a signal for cells to regulategK+. After removal of aldosterone, the increase ingK+ was preserved by subsequent incubation for 8 hr at pH 7.6 but abolished at pH 6.6. In the complete absence of aldosterone, incubation of cells at pH 8.0 for 20–28 hr raised pH
i
and doubledgK+.Using the patch-clamp technique, three types of K+-selective channels were identified, which had conductances of 24, 45 and 59 pS.Aldosterone had no effect on the conductance or open probability (P
o) of any of the three types of channels. However, the incidence of observing type II channels was increased from 4 to 22%. Type II channels were also found to be pH sensitive,P
o was increased by raising pH.These results indicate that prolonged aldosterone treatment raises pH
i
and increasesgK+ by promoting insertion of K+ channels into the cell membrane. Channel insertion is itself triggered by raising both pH
i
and increasing the activity of the Na+/K+ pump in early distal cells of frog kidney.
Present address: Department of Physiology, The University of Leeds, Leeds, LS2 9NQ, England 相似文献
9.
V. P. Skulachev 《Journal of bioenergetics and biomembranes》1989,21(6):635-647
The progress of bioenergetic studies on the role of Na+ in bacteria is reviewed. Experiments performed over the past decade on several bacterial species of quite different taxonomic positions show that Na+ can, under certain conditions, substitute for H+ as the coupling ion. Various primary Na+ pumps (
generators) are described, i.e., Na+-motive decarboxylases, NADH-quinone reductase, terminal oxidase, and ATPase. The
formed is shown to be consumed by Na+ driven ATP-synthase, Na+ flagellar motor, numerous Na+, solute symporters, and the methanogenesis-linked reverse electron transfer system. InVibrio alginolyticus, it was found that
, generated by NADH-quinone reductase, can be utilized to support all three types of membrane-linked work, i.e., chemical (ATP synthesis), osmotic (Na+, solute symports), and mechanical (rotation of the flagellum). InPropionigenum modestum, circulation of Na+ proved to be the only mechanism of energy coupling. In other species studied, the Na+ cycle seems to coexist with the H+ cycle. For instance, inV. alginolyticus the initial and terminal steps of the respiratory chain are Na+ - and H+-motive, respectively, whereas ATP hydrolysis is competent in the uphill transfer of Na+ as well as of H+. In the alkalo- and halotolerantBacillus FTU, there are H+ - and Na+-motive terminal oxidases. Sometimes, the Na+-translocating enzyme strongly differs from its H+-translocating homolog. So, the Na+-motive and H+-motive NADH-quinone reductases are composed of different subunits and prosthetic groups. The H+-motive and Na+-motive terminal oxidases differ in that the former is ofaa
3-type and sensitive to micromolar cyanide whereas the latter is of another type and sensitive to millimolar cyanide. At the same time, both Na+ and H+ can be translocated by one and the sameP. modestum ATPase which is of the F0F1-type and sensitive to DCCD. The sodium cycle, i.e., a system composed of primary
generator(s) and
consumer(s), is already described in many species of marine aerobic and anaerobic eubacteria and archaebacteria belonging to the following genera:Vibrio, Bacillus, Alcaligenes, Alteromonas, Salmonella, Klebsiella, Propionigenum, Clostridium, Veilonella, Acidaminococcus, Streptococcus, Peptococcus, Exiguobacterium, Fusobacterium, Methanobacterium, Methanococcus, Methanosarcin, etc. Thus, the sodium world seems to occupy a rather extensive area in the biosphere. 相似文献
10.
Clay JR 《Progress in biophysics and molecular biology》2005,88(1):59-90
The original papers of Hodgkin and Huxley (J. Physiol. 116 (1952a) 449, J. Physiol. 116 (1952b) 473, J. Physiol. 116 (1952c) 497, J. Physiol. 117 (1952d) 500) have provided a benchmark in our understanding of cellular excitability. Not surprisingly, their model of the membrane action potential (AP) requires revisions even for the squid giant axon, the preparation for which it was originally formulated. The mechanisms they proposed for the voltage-gated potassium and sodium ion currents, IK, and INa, respectively, have been superceded by more recent formulations that more accurately describe voltage-clamp measurements of these components. Moreover, the current-voltage relation for IK has a non-linear dependence upon driving force that is well described by the Goldman-Hodgkin-Katz (GHK) relation, rather than the linear dependence on driving force found by Hodgkin and Huxley. Furthermore, accumulation of potassium ions in the extracellular space adjacent to the axolemma appears to be significant even during a single AP. This paper describes the influence of these various modifications in their model on the mathematically reconstructed AP. The GHK and K+ accumulation results alter the shape of the AP, whereas the modifications in IK and INa gating have surprisingly little effect. Perhaps the most significant change in their model concerns the amplitude of INa, which they appear to have overestimated by a factor of two. This modification together with the GHK and the K+ accumulation results largely remove the discrepancies between membrane excitability of the squid giant axon and the Hodgkin and Huxley (J. Physiol. 117 (1952d) 500) model previously described (Clay, J. Neurophysiol. 80 (1998) 903). 相似文献
11.
Adam Bertl 《The Journal of membrane biology》1989,109(1):9-19
Summary The membrane of mechanically prepared vesicles ofChara corallina has been investigated by patch-clamp techniques. This membrane consists of tonoplast as demonstrated by the measurement of ATP-driven currents directed into the vesicles as well as by the ATP-dependent accumulation of neutral red. Addition of 1mm ATP to the bath medium induced a membrane current of about 3.2 mA·m–2 creating a voltage across the tonoplast of about –7 mV (cytoplasmic side negative). On excised tonoplast patches, currents through single K+-selective channels have been investigated under various ionic conditions. The open-channel currents saturate at large voltage displacements from the equilibrium voltage for K+ with limiting currents of about +15 and –30 pA, respectively, as measured in symmetric 250mm KCl solutions. The channel is virtually impermeable to Na+ and Cl–. However, addition of Na+ decreases the K+ currents. TheI–V relationships of the open channel as measured at various K+ concentrations with or without Na+ added are described by a 6-state model, the 12 parameters of which are determined to fit the experimental data. 相似文献
12.
Hanxing Wan Xiong Ying Chen Fenglian Zhang Jun Chen Fenglan Chu Zachary M. Sellers Feng Xu Hui Dong 《The Journal of biological chemistry》2022,298(5)
Although capsaicin has been studied extensively as an activator of the transient receptor potential vanilloid cation channel subtype 1 (TRPV1) channels in sensory neurons, little is known about its TRPV1-independent actions in gastrointestinal health and disease. Here, we aimed to investigate the pharmacological actions of capsaicin as a food additive and medication on intestinal ion transporters in mouse models of ulcerative colitis (UC). The short-circuit current (Isc) of the intestine from WT, TRPV1-, and TRPV4-KO mice were measured in Ussing chambers, and Ca2+ imaging was performed on small intestinal epithelial cells. We also performed Western blots, immunohistochemistry, and immunofluorescence on intestinal epithelial cells and on intestinal tissues following UC induction with dextran sodium sulfate. We found that capsaicin did not affect basal intestinal Isc but significantly inhibited carbachol- and caffeine-induced intestinal Isc in WT mice. Capsaicin similarly inhibited the intestinal Isc in TRPV1 KO mice, but this inhibition was absent in TRPV4 KO mice. We also determined that Ca2+ influx via TRPV4 was required for cholinergic signaling–mediated intestinal anion secretion, which was inhibited by capsaicin. Moreover, the glucose-induced jejunal Iscvia Na+/glucose cotransporter was suppressed by TRPV4 activation, which could be relieved by capsaicin. Capsaicin also stimulated ouabain- and amiloride-sensitive colonic Isc. Finally, we found that dietary capsaicin ameliorated the UC phenotype, suppressed hyperaction of TRPV4 channels, and rescued the reduced ouabain- and amiloride-sensitive Isc. We therefore conclude that capsaicin inhibits intestinal Cl- secretion and promotes Na+ absorption predominantly by blocking TRPV4 channels to exert its beneficial anti-colitic action. 相似文献
13.
Outward sodium and potassium cotransport in human red cells 总被引:7,自引:0,他引:7
Ricardo Garay Norma Adragna Mitzy Canessa Daniel Tosteson 《The Journal of membrane biology》1981,62(3):169-174
Summary This paper reports some kinetic properties of Na–K cotransport in human red cells. All fluxes were measured in the presence of 10–4 M ouabain. We measured Na and K efflux from cells loaded by the PCMBS method to contain different concentrations of these ions into a medium that contained neither Na nor K (MgCl2-sucrose substitution) in the absence and presence of furosemide. Furosemide inhibited 30–60% of the total efflux depending on the internal ion concentration and the individual subject. We took the furosemide-sensitive fluxes to be a measure of Na–K cotransport. The ratio of Na to K cotransport was 1 over the entire range of internal Na and K concentrations studied. When Na was substituted for K as the only internal cation, cotransport was maximally activated when the Na and K concentrations were between 20 and 90 mmol/liter cells. The concentration of internal Na required to produce half-maximal cotransport was about 13±4 mmol/liter cells (n=4), while the comparable concentration of K was somewhat lower. The activation curve was definitely sigmoid in character, suggesting that at least two Na ions are involved in the transport process. The maximum of Na–K cotransport was about 0.5±0.15 mmol/liter cells × hr (n=5); it had a flat maximum in the medium at about pH 7.0, decreasing in both the acid and alkaline sides. furosemide-resistant effluxes were found to be linear functions of internal Na and K concentrations and to yield rate coefficients of 0.019±0.002 hr–1 and 0.014±0.002 hr–1 (n=7), respectively. These values are of the same order of magnitude expected of ions moving across phospholipid bilayers.Charge de Recherches CNRS. 相似文献
14.
Johann S. Schwegler Wieland Steigner Almut Heuner Stefan Silbernagl 《The Journal of membrane biology》1990,117(3):243-251
Summary Confluent monolayers of the established opossum kidney cell line were exposed to NH4Cl pulses (20 mmol/liter) during continuous intracellular measurements of pH, membrane potential (PD
m
) and membrane resistance (R
m) in bicarbonate-free Ringer. The removal of extracellular NH4Cl leads to an intracellular acidification from a control value of 7.33±0.08 to 6.47±0.03 (n=7). This inhibits the absolute K conductance (g
K+), reflected by a decrease of K+ transference number from 71±3% (n=28) to 26±6% (n=5), a 2.6±0.2-fold rise ofR
m, and a depolarization by 24.2±1.5 mV (n=52). In contrast, intracellular acidification during a block ofg
K+ by 3 mmol/liter BaCl2 enhances the total membrane conductance, being shown byR
m decrease to 68±7% of control and cell membrane depolarization by 9.8±2.8 mV (n=17). Conversely, intracellular alkalinization under barium elevatesR
m and hyperpolarizes PD
m
. The replacement of extracellular sodium by choline in the presence of BaCl2 significantly hyperpolarizes PD
m
and increasesR
m, indicating the presence of a sodium conductance. This conductance is not inhibited by 10–4 mol/liter amiloride (n=7). Patch-clamp studies at the apical membrane (excised inside-out configuration) revealed two Na+-conductive channels with 18.8±1.4 pS (n=10) and 146 pS single-channel conductance. Both channels are inwardly rectifying and highly selective towards Cl–. The low-conductive channel is 4.8 times more permeable for Na+ than for K+. Its open probability rises at depolarizing potentials and is dependent on the pH of the membrane inside (higher at pH 6.5 than at pH 7.8). 相似文献
15.
J. Graf R. M. Henderson B. Krumpholz J. L. Boyer 《The Journal of membrane biology》1987,95(3):241-254
Summary The basic electrical properties of an isolated rat hepatocyte couplet (IRHC) system have been analyzed using classical techniques of epithelial electrophysiology, including measurement of electric potentials, resistances and intracellular ion activities. Applications of these techniques are discussed with respect to their limitations in small isolated cells. Mean intracellular and intracanalicular membrane potentials ranged from –23.7 to –46.7 and –4.3 to –5.9 mV, respectively. Membrane resistances were determined using an equivalent circuit analysis modified according to the geometry of the IRHC system. Resistances of the sinusoidal (basolateral) and canalicular (luminal) cell membranes and tight junctions averaged 0.15 and 0.78 G and 25m, respectively. The cells are electrically coupled via low resistance intercellular communications (58 M). Intracellular ion activities for Na+, K+ and Cl– averaged 12.2, 88.1 and 17.7 mmol/liter, respectively. The basolateral membrane potential reveals a permeability sequence ofP
K>P
Cl>P
Na. The luminal potential showed minimal dependence on changes in transjunctional ion gradients, indicating a poor ion selectivity of the paracellular pathway. The electrogenic (Na+–K)-ATPase contributes little to the luminal and cellular negative electric potential. Therefore, the luminal potential probably results from the secretion of impermeant ions and a Donnan distribution of permeant ions, a mechanism which provides the osmotic driving force for bile formation. By providing the unique opportunity to measure luminal potentials, this isolated hepatocyte system permits study of secretory mechanisms for the first time in a mammalian gland using electrophysiologic techniques. 相似文献
16.
17.
The effects of propyl paraben, an antimicrobial preservative, on voltage-dependent sodium current and myocardial ischemia-reperfusion injury were investigated in isolated adult rat cardiomyocytes. Whole cell voltage-clamp recording showed that propyl paraben reversibly blocked the voltage-gated sodium channel both in concentration- and voltage-dependent manners. Propyl paraben (500 microM but not 100 microM) significantly shifted the steady-state inactivation of the sodium channel toward the hyperpolarizing direction at the V(1/2) point. Consistent with the above result, the propidium iodide (PI) uptake test revealed that pretreatment with 500 microM but not 100 microM of propyl paraben significantly reduced cell death induced by 45 min of sustained ischemia followed by 15 h of reperfusion (42.37 +/- 7.01% of cell viability in control and 71.05 +/- 7.06% in the propyl paraben group), suggesting that propyl paraben can protect myocytes from ischemia-reperfusion injury. These results indicate a possible correlation between the inhibition of sodium current and cardioprotection against ischemia-reperfusion injury. 相似文献
18.
Sergei N. Orlov Therese J. Resink Jens Bernhardt Fritz R. Buhler 《The Journal of membrane biology》1992,129(2):199-210
Summary To identify ion transport systems involved in the maintenance of vascular smooth muscle cell volume the effects of incubation medium osmolality and ion transport inhibitors on the volume and 86Rb and 22Na transport in cultured smooth muscle cells from rat aorta (VSMC) have been studied. A decrease of medium osmolality from 605 to 180 mosm increased intracellular water volume from 0.6 to 1.3 l per 106 cells. Under isosmotic conditions, cell volume was decreased by ouabain (by 10%, P< 0.005) but was not influenced by bumetanide, furosemide, EIPA and quinidine. These latter compounds were also ineffective in cell volume regulation under hypotonic buffer conditions. Under hyperosmotic conditions, cell volume was decreased by bumetanide (by 7%, P<0.05) and by ethylisopropyl amiloride (by 13%, P< 0.005). Ouabain-sensitive 86Rb influx was decreased by 30–40% under hypoosmotic conditions. An increase in medium osmolality from 275 to 410 mosm resulted in an eightfold increase in bumetanide-inhibited 86Rb influx and 86Rb efflux. The (ouabain and bumetanide)-insensitive component of 86Rb influx was not dependent on the osmolality of the incubation medium. However (ouabain and bumetanide)-insensitive 86Rb efflux was increased by 1.5–2 fold in VSMC incubated in hypotonic medium. Ethylisopropyl amiloride-inhibited 22Na influx was increased by sixfold following osmotic-shrinkage of VSMC. The data show that both Na+/H+ exchange and Na+/K+/2Cl– cotransport may play a major role in the regulatory volume increase in VSMC. Basal and shrinkage-induced activities of Na+/K+/2Cl– cotransport in VSMC were similarly sensitive to inhibition by either staurosporin, forskolin, R24571 or 2-nitro4-carboxyphenyl N,N-diphenylcarbomate (NCDC). In contrast basal and shrinkage-induced Na+/K+/2Cl– cotransport were differentially inhibited by NaF (by 30 and 65%, respectively), suggesting an involvement of guanine nucleotide binding proteins in the volume-sensitive activity of this carrier. Neither staurosporin, forskolin, R24571 nor NCDC influenced shrinkage-induced Na+/H+ exchange activity. NaF increased Na+/H+ exchanger activity under both isosmotic and hyperosmotic conditions. These data demonstrate that different intracellular signalling mechanisms are involved in the volume-dependent activation of the Na+/K+/2Cl– cotransporter and the Na+/H+ exchanger.The authors gratefully acknowledge the financial support of the Swiss National Foundation, grant No. 3.817.087. Bernadette Weber is thanked for preparing the figures. 相似文献
19.
Gerasimos S. Filippatos W. Frank Hughes Renli Qiao J. Iasha Sznajder Bruce D. Uhal 《In vitro cellular & developmental biology. Animal》1997,33(3):195-200
Summary Active transport of sodium by pulmonary alveolar epithelial cells (AEC) is believed to be an important component of edema
clearance in the normal and injured lung. Data supporting this premise have come from measurements of sodium movement across
AEC monolayers or from perfused lung model systems. However, direct measurement of fluid flux across AEC monolayers has not
been reported. In the present work, AEC were studied with an experimental system for the measurement of fluid flux (Jv) across
functionally intact cell monolayers. Primary adult rat type II alveolar epithelial cells were cultured on 0.8 μm nuleopore
filters previously coated with gelatin and fibronectin. Intact monolayers were verified by high electrical resistance (> 1000
Θ) at 4–5 d of primary culture. At the same time interval, transmission electron microscopy revealed cells with type I cell-like
morphology throughout the monolayer. These were characterized by both adherens and tight junctional attachments. Fluid flux
across the monolayers was measured volumetrically over a period of 2 h in the presence of HEPES-buffered DMEM containing 3%
fatty acid-free bovine serum albumin. Flux (Jv) was inhibited 39% by 1 × 10−4
M ouabain (P < 0.01) and 27% by 5 × 10−4
M amiloride (P < 0.05). These data support the concept that AEC Na+/K+-ATPase and Na+ transport systems are important determinants of AEC transepithelial fluid movement in vitro. 相似文献
20.
Summary To study the physiological role of the bidirectionally operating, furosemide-sensitive Na+/K+ transport system of human erythrocytes, the effect of furosemide on red cell cation and hemoglobin content was determined in cells incubated for 24 hr with ouabain in 145mm NaCl media containing 0 to 10mm K+ or Rb+. In pure Na+ media, furosemide accelerated cell Na+ gain and retarded cellular K+ loss. External K+ (5mm) had an effect similar to furosemide and markedly reduced the action of the drug on cellular cation content. External Rb+ accelerated the Na+ gain like K+, but did not affect the K+ retention induced by furosemide. The data are interpreted to indicate that the furosemide-sensitive Na+/K+ transport system of human erythrocytes mediates an equimolar extrusion of Na+ and K+ in Na+ media (Na+/K+ cotransport), a 1:1 K+/K+ (K+/Rb+) and Na+/Na+ exchange progressively appearing upon increasing external K+ (Rb+) concentrations to 5mm. The effect of furosemide (or external K+/Rb+) on cation contents was associated with a prevention of the cell shrinkage seen in pure Na+ media, or with a cell swelling, indicating that the furosemide-sensitive Na+/K+ transport system is involved in the control of cell volume of human erythrocytes. The action of furosemide on cellular volume and cation content tended to disappear at 5mm external K+ or Rb+. Thein vivo red cell K+ content was negatively correlated to the rate of furosemide-sensitive K+ (Rb+) uptake, and a positive correlation was seen between mean cellular hemoglobin content and furosemide-sensitive transport activity. The transport system possibly functions as a K+ and waterextruding mechanism under physiological conditiosin vivo. The red cell Na+ content showed no correlation to the activity of the furosemide-sensitive transport system. 相似文献